HUMAN-IGG MONOCLONAL AUTOANTIBODIES AGAINST MUSCLE ACETYLCHOLINE-RECEPTOR - DIRECT EVIDENCE FOR CLONAL HETEROGENEITY OF THE ANTISELF HUMORAL RESPONSE IN MYASTHENIA-GRAVIS

Human hybridomas were established from myasthenia gravis (MG) patients and screened using a fast and sensitive cell ELISA with the rhabdomyo sarcoma cell line TE671. In a first series of 14 fusions using a stand ard protocol, 36 positive clones were detected and maintained for thre e passages. The number of clones in each fusion was correlated with in vivo titers of anti-acetylcholine receptor (AChR) autoantibodies. In a second series of four experiments, fusions were immediately followed by cell plating under limiting dilution conditions ('fusion cloning') providing eight stable hybridomas. These hybridomas produced monoclon al antibodies (mAbs) of IgG isotype reactive with TE671 cells, but not with AChR in solution using the radioimmunoprecipitation assay. Fine analysis of antigen specificity of these mAbs was performed using soli d-phase ELISA against purified AChR from Torpedo (T-AChR) and immunobl ot against recombinant chimaeric human AChR produced in bacteria. Five of the eight mAbs derived from the few patients whose antibodies show ed cross-reactivity with T-AChR reacted against T-AChR. Of these five mAbs, two also reacted against chimaeric human AChR by immunoblotting. Furthermore, at least one of these two mAbs was capable of inducing a ntigenic modulation of labeled AChR with [(125)Il alpha-bungarotoxin f rom the surface of TE671 cells. These mAbs provide useful tools to exp lore the molecular basis of the structural and functional heterogeneit y of the humoral anti-AChR response in myasthenia gravis.